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bioRxiv · 10.1101/2020.11.05.370742

Developing a broadly applicable pyrF-based genetic manipulation system in Acinetobacter baumannii

Xu, R.·Gao, C.·Wu, S.·Su, M.·Sun, C.·Jia, X.·Wang, R.
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Abstract

Acinetobacter baumannii is an emergency pathogenic bacterium for its multidrug-resistance and high mortality rates after infection. In-depth genetic analysis of A. baumannii virulence and drug-resistant genes is highly desirable. Existing methods for genetic manipulation of A. baumannii mainly rely on the use of antibiotic as the selectable marker, and the sacB/sucrose as the counter-selectable marker, which is inconvenient and inappropriate for all research of A. baumannii. Based on the highly conserved pyrF gene and its conserved 500bp-flanking sequence, we quickly and easily generated the pyrF-deleted mutants as the uracil auxotrophic host strain in three model strains and 11 clinical strains. The pyrF-carried vectors constructed for gene editing with pyrF/5-FOA as the counter-selection were conveniently and time-saving in these pyrF-deleted mutants. Utilizing the pyrF-based genetic manipulation system, we easily and efficiently modified the cas gene and CRISPR sequence of I-F CRISPR-Cas system in A. baumannii AYE, and detected the CRISPR interference and adaptation in these mutants. In summary, the pyrF-based genetic manipulation system could be broadly applicable used for efficiently maker-less gene editing in most A. baumannii strains.

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Xu, R., Gao, C., Wu, S., Su, M., Sun, C., Jia, X., Wang, R.. 2020-11-06. Developing a broadly applicable pyrF-based genetic manipulation system in Acinetobacter baumannii. https://doi.org/10.1101/2020.11.05.370742

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